This proposal represents a request for continued support of our ongoing project exploring receptor-ligand interactions and the thermodynamics of ligand binding to biological receptors. The overall objectives are aimed at developing and applying methods of free energy simulations to ligand binding thermodynamics, docking and receptor-ligand interaction modeling. Specific efforts are directed to the development of a hierarchy of methods that provide suitable tools for high-throughput screening, as well as detailed ligand refinement techniques employing accurate atomic force fields and adequate sampling. The primary goals of the current proposal are (i) the development, implementation and assessment of flexible receptor - flexible ligand docking approaches, augmenting the previously developed CDOCKER pipeline with flexible receptor sampling employing torsion angle molecular dynamics and NMR-derived ensembles for flexible RNA-based therapeutic targets, and (ii) the development of free energy based simulation methods to enable multi-site lambda dynamics free energy calculations. The former method development will be driven by collaborative studies to identify and develop mimics of transcriptional activation domains (TADs) based on amphipathic small molecules, initially targeting activation through interactions with KIX domains of the CREB binding protein and utilizing chemistry focused around isoxazolidine based compounds. Additional driving biomedical questions concerning the development of small molecule inhibitors for RNA targets will be addressed through collaborative studies and aim at establishing robust in silico screening approaches of small molecules toward RNA receptors such as HIV TAR. Finally, collaborative studies directed toward the discovery and refinement of potential cancer therapeutics directed toward inhibition of Golgi 1-mannosidase will be pursued utilizing the novel multi-site lambda dynamics methods developed through this project. PUBLIC HEALTH RELEVANCE: New computer based docking and free energy simulation methods will be developed to address the discovery and refinement of small molecule transcriptional activators for the control transcriptional activation, small molecule inhibitors targeted for RNA-based receptors in HIV TAR, and cancer therapeutics that target inhibition of the glycosylation enzyme Golgi 1-mannosidase.